The Autism-Related Protein CHD8 Cooperates with C/EBPβ to Regulate Adipogenesis

Highlights

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CHD8 directly regulates C/EBPα and PPARγ during adipogenesis

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CHD8 interacts with C/EBPβ and promotes its transactivity

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CHD8 ablation in preadipocytes results in a markedly reduced fat mass

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CHD8 is essential for not only neuronal development but also adipose development

Summary

The gene encoding the chromatin remodeler CHD8 is the most frequently mutated gene in individuals with autism spectrum disorder (ASD). Heterozygous mutations in CHD8 give rise to ASD that is often accompanied by macrocephaly, gastrointestinal complaints, and slender habitus. Whereas most phenotypes of CHD8 haploinsufficiency likely result from delayed neurodevelopment, the mechanism underlying slender habitus has remained unknown. Here, we show that CHD8 interacts with CCAAT/enhancer-binding protein β (C/EBPβ) and promotes its transactivation activity during adipocyte differentiation. Adipogenesis was impaired in Chd8-deleted preadipocytes, with the upregulation of C/EBPα and peroxisome-proliferator-activated receptor γ (PPARγ), two master regulators of this process, being attenuated in mutant cells. Furthermore, mice with CHD8 ablation in white preadipocytes had a markedly reduced white adipose tissue mass. Our findings reveal a mode of C/EBPβ regulation by CHD8 during adipogenesis, with CHD8 deficiency resulting in a defect in the development of white adipose tissue.

Introduction

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a high prevalence (∼2% of the global population) (Schaaf and Zoghbi, 2011, Taylor et al., 2013). Recent exome-sequencing studies of individuals with ASD have identified many mutations, with the gene encoding the chromatin remodeler CHD8 (chromodomain helicase DNA-binding protein 8) being the most frequent site of such mutations (Neale et al., 2012, O’Roak et al., 2012a, O’Roak et al., 2012b, Talkowski et al., 2012). Many studies of CHD8 have thus focused on its role in neurogenesis and brain development (Cotney et al., 2015, Sugathan et al., 2014). CHD8 is expressed not only in neuronal tissues but also in many other organs, where it may also play an important developmental role (Ishihara et al., 2006, Nishiyama et al., 2009). Indeed, individuals with CHD8 mutations manifest not only ASD but also macrocephaly, distinct facial characteristics, gastrointestinal complaints, and a tall and slender habitus. Although psychological symptoms, macrocephaly, and gastrointestinal complaints resulting from CHD8 haploinsufficiency are likely attributable to defective neuronal development and associated responses, the mechanism by which CHD8 mutations give rise to slenderness has remained unclear (Bernier et al., 2014, Katayama et al., 2016). Such slenderness is an unexpected characteristic, given that ASD patients in general tend to be obese as a result of food selectivity, gastrointestinal symptoms, reduced physical activity, and medication use (Curtin et al., 2010, Curtin et al., 2014, Zheng et al., 2017). These observations suggest that CHD8 might also contribute to regulation of metabolism or adipogenesis.

CHD8 is a member of the CHD family of enzymes that belong to the SNF2 superfamily of ATP-dependent chromatin remodelers and are defined by the presence of two chromodomains: a helicase/ATPase domain and a DNA-binding domain (Hall and Georgel, 2007, Marfella and Imbalzano, 2007). Several members of this family play important roles during development and are implicated in various human diseases (Ho and Crabtree, 2010). For example, CHD1 is thought to contribute to the pluripotency of embryonic stem cells by maintaining an open chromatin state (Gaspar-Maia et al., 2009). Similarly, CHD5 is a key regulator of neurogenesis and plays a dual role in the activation of neuronal genes and the repression of Polycomb target genes (Egan et al., 2013). The CHD5 gene resides within a region of human chromosome 1p36 that shows loss of heterozygosity in individuals at high risk for the development of neuroblastoma (Bagchi et al., 2007). CHD7 is also required for neurogenesis and neural crest formation as a result of its direct binding to promoter or enhancer regions of fate-controlling transcription factor genes and consequent opening of chromatin structure (Bajpai et al., 2010, Feng et al., 2013). De novo mutations of human CHD7 give rise to CHARGE syndrome, a condition characterized by malformation of various organs (Vissers et al., 2004).

CHD8 was originally identified as a negative regulator of the Wnt/β-catenin signaling pathway (Nishiyama et al., 2012, Sakamoto et al., 2000). CHD8 generates two alternatively spliced transcripts that encode a 280-kDa full-length protein (CHD8L) or a 110-kDa protein (CHD8S) that contains only the NH2-terminal chromodomain (Ishihara et al., 2006, Nishiyama et al., 2009). Mutations identified in individuals with ASD are distributed throughout the CHD8 locus, with some expected to result in the loss of both CHD8 isoforms and others to affect only CHD8L. To recapitulate this situation, we generated two independent lines of mutant mice deficient in both CHD8 isoforms (ΔSL) or only CHD8L (ΔL), and we found that both heterozygous mutant strains manifest ASD-like behavioral characteristics (Katayama et al., 2016). CHD8 was also shown to regulate the expression of ASD-related neurodevelopmental genes in neurons, especially those targeted by RE1-silencing transcription factor (REST), which suppresses the transcription of many neuronal genes during development.

Adipogenesis is the strictly controlled cellular process by which preadipocytes differentiate into mature adipocytes. The expression of preadipogenic transcription factors such as CCAAT/enhancer-binding protein β (C/EBPβ) and C/EBPδ is induced during the early phase of adipogenesis and is followed by that of C/EBPα and peroxisome-proliferator-activated receptor γ (PPARγ), both of which are master regulators of adipogenesis. C/EBPα and PPARγ activate the expression of multiple adipogenic genes whose products mediate the final maturation process (Siersbæk et al., 2012). In response to adipogenic stimulation, chromatin structure undergoes substantial remodeling, resulting in the activation of a complex network of these transcription factors (Rosen and MacDougald, 2006). The mechanistic basis of such chromatin remodeling during adipogenesis has remained largely unknown, however.

We now show that CHD8 is essential for adipogenesis and the development of white adipose tissue (WAT). We found that CHD8 cooperates with C/EBPβ to regulate transactivation of the genes for C/EBPα and PPARγ during adipogenesis. Generation of mice in which Chd8 is deleted specifically in mesenchymal stem cells revealed that these animals have a markedly reduced adipose tissue mass. Our results thus indicate that CHD8 plays an essential role not only in neuronal development but also in adipogenesis.

Results

CHD8 Is Required for Adipogenesis

To investigate the role of CHD8 during adipogenesis, we established immortalized Chd8LF/F preadipocyte lines from inguinal WAT (iWAT) of 4-week-old mice and interscapular brown adipose tissue (BAT) of 2-day-old mice that were homozygous for a floxed (F) allele of Chd8 (Figure 1A). The cells were infected with a retrovirus encoding Cre recombinase to generate Chd8L−/− preadipocytes or with a control retrovirus encoding EGFP. We confirmed that Cre inactivated almost all floxed alleles in Chd8LF/F preadipocytes (Figures 1B and 1D). Ablation of Chd8 in preadipocytes resulted in a pronounced defect in adipogenesis in response to treatment with the combination of 3-isobutyl-1-methylxanthine, dexamethasone, and insulin (MDI) (Figures 1C and 1E). Consistent with this impairment of adipogenesis, RT-PCR and real-time PCR analysis showed that the induction of adipogenic marker genes such as Cebpa, Fabp4, Fas, Glut4, and Pparg was significantly attenuated in CHD8-deficient cells (Figure 1F). We also isolated preadipocytes from iWAT of wild-type (WT) or Chd8 heterozygous mutant (Chd8+/ΔL) mice (Katayama et al., 2016) (Figure 1G). Exposure of these cells to MDI revealed that the ability of Chd8+/ΔLpreadipocytes to undergo adipogenic differentiation was impaired compared with that of WT cells (Figure 1H). The induction of adipogenic marker genes was also attenuated in Chd8+/ΔL cells compared with WT cells (Figure 1I). Furthermore, we examined the effect of short hairpin RNA (shRNA)-mediated depletion of CHD8 on adipogenesis in the white preadipocyte cell line 3T3-L1 (Figure 1J). Adipogenesis and adipogenic gene expression were attenuated in these cells in a manner related to the extent of CHD8 depletion (Figures 1K and 1L), indicating that CHD8 is also important for adipocytic differentiation of 3T3-L1 cells. Together, these results showed that CHD8 plays an essential role in adipogenesis in vitro.

To explore the mechanism underlying the impairment of adipogenesis in CHD8-deficient preadipocytes, we compared the expression of several adipogenic transcription factors between Chd8L−/− and Chd8LF/F preadipocytes during adipogenesis. We first showed that the expression of CHD8 was markedly upregulated as early as day 1 or 2 after the onset of adipogenic induction in 3T3-L1 cells or in brown preadipocytes isolated from WT mice (Figures 2A and 2B ). Furthermore, ablation of CHD8 did not substantially affect the steady-state expression levels of preadipogenic genes such as Cebpb, Cebpd, Krox20, Pparg1, and Pref1 in Chd8LF/F preadipocytes or 3T3-L1 preadipocytes (Figures 2C and 2D). Whereas C/EBPβ, which functions upstream of Cebpa and Pparg, was induced normally at both mRNA and protein levels by MDI treatment in Chd8L−/−preadipocytes (Figures 2E and 2F), the deletion of Chd8 essentially prevented the activation of Cebpa and Pparg expression (Figures 2G and 2H).

We next investigated whether CHD8 might be associated with the promoter or enhancer regions of Cebpa or Pparg in preadipocytes of iWAT from WT mice using chromatin immunoprecipitation (ChIP) analysis (Guo et al., 2012, Wang et al., 2013). Endogenous CHD8 was indeed found to be associated with the promoter and enhancer regions of both Cebpa (Figure 3A) and Pparg (Figure 3B). No substantial association of CHD8 with the promoter region of Runx2 (control) was detected (Figure 3A). CHD8 binding to Cebpa and Pparg1 promoter regions was not affected by exposure of the cells to MDI, whereas that to the enhancer regions of both genes and to the Pparg2 promoter region was upregulated (Figure 3C). Deletion of Chd8 essentially abolished CHD8 association with the promoter and enhancer regions of Cebpa and Pparg (Figure 3D). Consistent with these results, forced expression of CHD8 increased Cebpa and Pparg2 promoter activities in a concentration-dependent manner (Figure 3E). We also infected Chd8L−/− preadipocytes with a retrovirus encoding PPARγ2. Such ectopic expression of PPARγ2 fully rescued adipogenesis in the CHD8-deficient preadipocytes (Figure 3F). Together, these results thus suggested that CHD8 interacts with the promoter and enhancer regions of Cebpa and Pparg and promotes transactivation of these genes during the early phase of adipogenesis.

CHD8 Interacts with C/EBPβ and Regulates Adipogenic Gene Induction

Given that C/EBPβ is an upstream transcription factor of C/EBPα and PPARγ, we speculated that CHD8 might function cooperatively with C/EBPβ. Consistent with this notion, co-immunoprecipitation analysis revealed that FLAG-epitope-tagged C/EBPβ interacted with endogenous CHD8 in HEK293T cells and that endogenous C/EBPβ interacted with endogenous CHD8, but not with cyclin A (a nuclear protein examined as a negative control), in MDI-treated 3T3-L1 cells (Figures 4A–4C). Furthermore, luciferase reporter assays showed that CHD8 and C/EBPβ acted cooperatively to activate Cebpa and Pparg2 promoters (Figure 4D). FAIRE (formaldehyde-assisted isolation of regulatory elements) analysis revealed a reduced extent of chromatin opening at the promoters of both Cebpa and Pparg in Chd8-deleted preadipocytes exposed to MDI compared with MDI-treated control cells (Figure 4E).

We next investigated whether the association of CHD8 with chromatin at Cebpa and Pparg loci is dependent on C/EBPβ. We depleted 3T3-L1 cells of C/EBPβ by shRNA-mediated RNAi and then examined whether the localization of CHD8 was affected. The amount of C/EBPβ protein in cells expressing the corresponding shRNA and exposed to MDI for 2 days was reduced to ∼30% of that in control cells expressing an EGFP shRNA, whereas the abundance of CHD8 was not affected by C/EBPβ knockdown (Figure 4F). The binding of C/EBPβ to the Pparg2 promoter was also correspondingly attenuated in the C/EBPβ-deficient cells (Figure 4G). ChIP analysis revealed that the extent of CHD8 binding to the Cebpa and Pparg2 promoters in C/EBPβ-depleted cells was reduced compared with that in control cells (Figure 4H). In contrast, CHD8 binding to the Pparg1 promoter, with which C/EBPβ does not interact (Siersbæk et al., 2011), was unaffected by the depletion of C/EBPβ (Figure 4H), suggesting that the attenuation of CHD8 binding to the Cebpa and Pparg2 promoters was specific. These results thus indicate that the genomic localization of CHD8 is dependent, at least in part, on C/EBPβ. Conversely, the localization of C/EBPβ also appeared to be dependent on CHD8 (Figure 4I), suggesting that CHD8 and C/EBPβ bind to genomic sites in a mutually dependent manner (Figure 4J).

CHD8 Colocalizes with C/EBPβ at the Genome-wide Level

To determine the extent of colocalization of CHD8 and C/EBPβ at the genome-wide level, we performed ChIP and sequencing (ChIP-seq) analysis with antibodies to CHD8 in 3T3-L1 cells at 4 hr after the onset of MDI treatment. The resulting data were then integrated with published ChIP-seq and DNaseI-hypersensitive site (DHS)-sequencing profiles (Siersbæk et al., 2011). A total of 71,830 CHD8 peaks were detected, and these peaks were annotated on the basis of genomic features. Most of the peaks localized to intergenic (34%), intron (26%), and transcription start site (TSS; 25%) regions (Figure 5A). Approximately half of genes (53%) had one or more CHD8 peaks within ±50 kbp from the TSS (Figure 5B). We found that 16% of CHD8 peaks overlapped with DHSs, and that 20% of these peaks colocalized with C/EBPβ peaks, including those associated with typical C/EBPβ target genes such as Cebpa, Klf5, Pparg2, and Wnt10b (Figures 5C–5E). The vast majority of CHD8 peaks that overlapped with C/EBPδ, the glucocorticoid receptor (GR), signal transducer and activator of transcription 5A (STAT5A), or the retinoid X receptor (RXR) were also occupied by C/EBPβ (Figure 5F), although the colocalization rate of CHD8 with these four transcription factors was low (Figure 5D). These results thus indicated that CHD8 mainly colocalizes with C/EBPβ at open chromatin sites and that the two proteins regulate the expression of adipogenic genes in a cooperative manner.

We next performed RNA sequencing (RNA-seq) analysis in CHD8-depleted and control 3T3-L1 cells at 12 h after the onset of MDI treatment and then integrated the resulting data with those of our ChIP-seq analysis with antibodies to CHD8. RNA-seq analysis revealed that the expression of 11% of genes was upregulated and that of 11% of genes was downregulated in the CHD8-depleted cells (Figure 5G; Data S1). We also examined our ChIP-seq and RNA-seq data for 43 key adipogenesis-associated genes highlighted in recent reviews (Siersbæk et al., 2012, Stephens, 2012). Gene set enrichment analysis (GSEA) revealed a significant decrease in the expression of pro-adipogenic genes in the cells depleted of CHD8 compared with control cells, whereas anti-adipogenic genes did not show enrichment (Figure 5H). ChIP-seq analysis revealed that all 43 adipogenic genes had CHD8 peaks, with these peaks in 39 of the genes overlapping with DHSs, whereas 47% of all genes did not have CHD8 peaks (Figures 5B and 5I). We identified 19 genes at which CHD8 and C/EBPβ colocalized, including Cebpa and Pparg, suggesting that the two proteins cooperate to promote the expression of these adipogenic genes. Collectively, our results thus suggested that CHD8 regulates the expression of many adipogenic genes in association with C/EBPβ and thereby promotes adipogenesis.

CHD8 Is Essential for WAT Development

To investigate CHD8 function in vivo, we generated Prx1-Cre/Chd8LF/F mice by crossing Chd8LF/F mice with Prx1-Cre mice. Expression of Prx1-Cre in adipose tissue is limited to preadipocytes of iWAT, rendering Prx1-Cre-dependent recombination useful for depot-restricted genetic manipulation (Figure 6A) (Krueger et al., 2014, Sanchez-Gurmaches et al., 2015). To examine whether Prx1-Cre-dependent deletion of Chd8 inhibits adipogenesis, we fed Prx1-Cre/Chd8LF/F mice and Chd8LF/F (control) mice either a low-fat diet (LFD) or a high-fat diet (HFD) for 11 weeks beginning at 4 weeks of age. Although body weight was similar in Prx1-Cre/Chd8LF/F mice and Chd8LF/F mice fed either diet (Figure 6B), the gross appearance of iWAT was substantially affected in the former animals (Figures 6C, 6D, and S1A). Computed tomography (CT) also revealed that subcutaneous fat mass was markedly reduced in Prx1-Cre/Chd8LF/F mice fed either diet compared with control mice, whereas visceral fat mass did not differ between the two genotypes (Figures 6E and 6F). Although gonadal WAT (gWAT) weight and interscapular BAT weight were similar in Prx1-Cre/Chd8LF/F mice and Chd8LF/F mice (Figures 6G, 6H, and S1B), iWAT weight was significantly smaller in the former animals fed either an LFD or HFD (Figure 6I).

CHD8 Controls Adipogenesis and Adipocyte Hypertrophy in Vivo

Histological analysis showed that the area of vascular tissue in iWAT was increased in Prx1-Cre/Chd8LF/F mice fed an LFD compared with control animals, whereas adipocyte size did not differ between the two genotypes (Figure 6J). In contrast, the size of adipocytes in iWAT of Prx1-Cre/Chd8LF/F mice fed an HFD was reduced compared with that in control mice (Figures 6K and 6L). The amounts of mRNAs for preadipogenic genes were increased, whereas those of mRNAs for adipogenic genes were greatly reduced, in iWAT of Prx1-Cre/Chd8LF/F mice fed an LFD compared with control mice (Figure 6M), suggesting that the smaller fat mass of Prx1-Cre/Chd8LF/F mice maintained on such a diet was attributable to reduced adipogenesis. The expression levels of adipogenic genes in iWAT of Prx1-Cre/Chd8LF/F mice fed an HFD were also reduced compared with those in control mice, suggesting that the smaller fat mass of the mutant animals fed an HFD is attributable to reduced adipogenesis and adipocyte hypertrophy (Figure 6N). Consistent with these observations, preadipocytes isolated from Prx1-Cre/Chd8LF/Fmice were impaired in the ability to form mature adipocytes (Figures S1C–S1E).

To examine whether CHD8 is essential for HFD-induced adipocyte hypertrophy, we generated CAG-CreERT2/Chd8LF/F mice by crossing Chd8LF/F mice with CAG-CreERT2 mice. We then administered tamoxifen to 6-week-old CAG-CreERT2/Chd8LF/F mice by oral gavage for five consecutive days and confirmed that almost all floxed alleles were inactivated in preadipocytes derived from iWAT of the treated animals (Figure 7A). Tamoxifen-treated CAG-CreERT2/Chd8LF/F mice fed an HFD beginning after the end of tamoxifen administration showed a significantly reduced body weight, fat mass, and WAT weight compared with Chd8LF/F (control) mice (Figures 7B–7F). Moreover, the size of adipocytes (Figure 7G) as well as the expression levels of adipogenic genes (Figure 7H) in iWAT of tamoxifen-treated CAG-CreERT2/Chd8LF/F mice fed an HFD were reduced compared with those in control mice. Collectively, these results suggested that CHD8 is required in a cell-autonomous manner for both adipogenesis and adipocyte hypertrophy during WAT development.

Discussion

We have shown that CHD8 interacts with C/EBPβ and positively regulates C/EBPα and PPARγ gene expression during adipogenesis. Deletion of Chd8 specifically in preadipocytes of iWAT resulted in a marked reduction in corresponding adipose tissue mass, suggesting that impairment of adipogenesis occurs in a cell-autonomous manner. Although individuals with ASD in general tend to be overweight or prone to obesity (Curtin et al., 2010, Curtin et al., 2014, Zheng et al., 2017), those with CHD8 mutations tend to be slender (Bernier et al., 2014). We now conclude that this latter phenotype is not the result of a neurodevelopmental defect but is instead due to impairment of adipogenesis.

Adipogenesis is controlled by a complex network of transcription factors that mediate the differentiation of preadipocytes into mature adipocytes (Rosen and MacDougald, 2006). In the early phase of adipogenesis, multiple adipogenic signals activate a variety of preadipogenic transcription factors, including C/EBPβ, C/EBPδ, GR, and STAT5A, that colocalize at transcriptional regulatory regions of the genome and coordinate the assembly of functional “hotspots,” resulting in transcriptional activation of target genes such as Cebpa and Pparg via DNA looping and interaction with promoter regions (Siersbæk et al., 2014). Our results now identify CHD8 as an important coactivator of C/EBPβ in this process, with the extent of CHD8 association with transcriptional regulatory regions of Cebpa and Pparg increasing in response to adipogenic stimulation. Our results thus suggest that adipogenic stimulation induces binding of CHD8 to transcription regulatory regions or to preadipogenic transcription factors, including C/EBPβ, and that such association is required for proper adipogenesis. Given that dynamic chromatin loops that link promoter and enhancer regions are implicated in regulation of the expression of specific genes during adipogenesis (Siersbaek et al., 2017), we speculate that CHD8 might play a role in mediating such interactions. The molecular mechanism underlying these promoter-enhancer interactions and the identity of transcription factors that may function with CHD8 remain to be elucidated.

An increase in both adipocyte cell size (hypertrophy) and number (hyperplasia) is required for adipose tissue growth. We have shown that both the formation of adipocytes (adipogenesis) and subsequent adipocyte hypertrophy were abrogated in WAT of CHD8-deficient mice (Figure 6), whereas cells in which Chd8 was deleted by retroviral expression of Cre recombinase in vitro showed only a defect in adipogenesis (Figure 1E). One possible explanation for these findings is that although CHD8 is indispensable for both adipogenesis and adipocyte hypertrophy, Chd8L deletion due to Cre expression driven by the Prx1 promoter in vivo is not complete during adipogenesis, with the result that some cells undergo delayed Chd8 deletion after adipogenesis and then manifest a defect in adipocyte hypertrophy. In contrast, the efficiency of Chd8L deletion due to retrovirus-mediated Cre expression in Chd8LF/F preadipocytes in vitro appears to be ∼100%, with the resulting almost complete block of adipogenesis precluding manifestation of the effect of CHD8 loss on adipocyte hypertrophy (Figure S2).

Adipocytes play a central role in energy storage, lipid metabolism, and glucose homeostasis (Rosen and Spiegelman, 2006). Given that excessive adipocyte hypertrophy and hyperplasia lead to obesity and obesity-related disorders such as type 2 diabetes, hypertension, hyperlipidemia, and arteriosclerosis, a full understanding of adipogenesis is important for elucidation of the mechanisms underlying these various conditions (Muir et al., 2016). Our identification of the role of CHD8 in this process provides a basis for the development of potential treatments for obesity and type 2 diabetes.

Acknowledgments

We thank T. Kitamura and T. Akagi for providing vectors and DNA; N. Nishimura, K. Tsunematsu, and other laboratory members for technical assistance; and A. Ohta for help in preparation of the manuscript. This study was funded in part by KAKENHI grants (2522130, 26640080, and 17H06301) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. It was also supported in part by a Japan Society for the Promotion of Science (JSPS) fellowship (13J04520) to Y. Kita.

Author Contributions

Y. Kita planned and performed all experiments. Y. Katayama created Chd8+/ΔL and Chd8LF/F mice, performed experiments, and provided intellectual support. T. Shiraishi, K.M., and T.O. assisted with experiments. Y. Ohkawa, M. Suyama and T. Sato performed sequencing and data analysis. M.N. and M. Shirane provided materials and intellectual support. Y. Oike provided intellectual support. K.I.N. coordinated the study, oversaw the results and wrote the manuscript. All authors discussed the results and commented on the manuscript.